Bottom Line:
We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement.These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression.However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. hathcock@exchange.nih.gov

ABSTRACTT cell development occurs in the thymus and is critically dependent on productive TCRβ rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (β checkpoint), which is uniquely permissive for V-DJβ recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRβ rearrangement and pre-TCR expression enforces suppression of TCRβ rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRβ product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the β checkpoint, would suppress all V-DJβ rearrangement. We previously reported that premature CD28 signaling in murine CD4(-)CD8(-) (DN) thymocytes supports differentiation of CD4(+)CD8(+) (DP) cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRβ rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

pone-0020639-g001: Analysis of thymic development and BrdU incorporation in mice undergoing pre-TCR-dependent or CD28/B7-dependent development.(A) Thymocytes from B6, CD28/B7/B6, CD3εKO, and CD28/B7/CD3εKO mice were analyzed for CD4-CD8 subsets (CD4-PE and CD8-Al594) or for lineage negative DN subsets (CD25-PE and CD44-APC). Thymus numbers are to the right of these figures. Staining profiles are representative of >40 mice of each genotype analyzed. (B) Thymi were harvested from mice injected with BrdU, lineage negative cells were gated on the indicated DN subsets, and analyzed for BrdU incorporation. These histograms show the percentage BrdU+ cells present in all DN3 cells, early DN3 (CD25hi) cells, or late DN3 (CD25low) cells from B6 or CD28/B7/CD3εKO mice. This experiment is representative of three independent experiments performed.

Mentions:
We previously reported that during conventional thymic development, CD28-B7 interactions prior to pre-TCR expression are limited and that expression of both CD28 and B7-2 as transgenes (CD28/B7) beginning early in DN cells substantially altered thymic development, bypassing the requirement for pre-TCR at the β checkpoint [7]. In wild-type B6 mice undergoing pre-TCR-dependent development, the DN3 to DN4 transition is rate limiting as cells rearrange and test for functional TCRβ; this developmental check point is reflected by a DN3 to DN4 ratio of 3.2+/−0.3 (n = 28). In contrast, in CD28/B7/B6 thymocytes the DN3 to DN4 ratio is substantially reduced (0.2+/−0.02, n = 15). In addition, expression of CD28/B7 transgenes on a CD3εKO background (CD28/B7/CD3εKO) reversed the developmental arrest observed in pre-TCR deficient CD3εKO mice and allowed the efficient generation of DP cells (Figure 1A) [8], [9]. The DN3 to DN4 ratio in CD28/B7/CD3εKO thymocytes (0.2+/−0.03, n = 9) is similar to that for CD28/B7/B6. The reversal of the DN3 to DN4 ratios observed in mice expressing transgenic CD28/B7 suggests that early CD28 signaling both replaces the requirement for pre-TCR function and accelerates development through the β checkpoint.

pone-0020639-g001: Analysis of thymic development and BrdU incorporation in mice undergoing pre-TCR-dependent or CD28/B7-dependent development.(A) Thymocytes from B6, CD28/B7/B6, CD3εKO, and CD28/B7/CD3εKO mice were analyzed for CD4-CD8 subsets (CD4-PE and CD8-Al594) or for lineage negative DN subsets (CD25-PE and CD44-APC). Thymus numbers are to the right of these figures. Staining profiles are representative of >40 mice of each genotype analyzed. (B) Thymi were harvested from mice injected with BrdU, lineage negative cells were gated on the indicated DN subsets, and analyzed for BrdU incorporation. These histograms show the percentage BrdU+ cells present in all DN3 cells, early DN3 (CD25hi) cells, or late DN3 (CD25low) cells from B6 or CD28/B7/CD3εKO mice. This experiment is representative of three independent experiments performed.

Mentions:
We previously reported that during conventional thymic development, CD28-B7 interactions prior to pre-TCR expression are limited and that expression of both CD28 and B7-2 as transgenes (CD28/B7) beginning early in DN cells substantially altered thymic development, bypassing the requirement for pre-TCR at the β checkpoint [7]. In wild-type B6 mice undergoing pre-TCR-dependent development, the DN3 to DN4 transition is rate limiting as cells rearrange and test for functional TCRβ; this developmental check point is reflected by a DN3 to DN4 ratio of 3.2+/−0.3 (n = 28). In contrast, in CD28/B7/B6 thymocytes the DN3 to DN4 ratio is substantially reduced (0.2+/−0.02, n = 15). In addition, expression of CD28/B7 transgenes on a CD3εKO background (CD28/B7/CD3εKO) reversed the developmental arrest observed in pre-TCR deficient CD3εKO mice and allowed the efficient generation of DP cells (Figure 1A) [8], [9]. The DN3 to DN4 ratio in CD28/B7/CD3εKO thymocytes (0.2+/−0.03, n = 9) is similar to that for CD28/B7/B6. The reversal of the DN3 to DN4 ratios observed in mice expressing transgenic CD28/B7 suggests that early CD28 signaling both replaces the requirement for pre-TCR function and accelerates development through the β checkpoint.

Bottom Line:
We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement.These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression.However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

Affiliation:
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. hathcock@exchange.nih.gov

ABSTRACTT cell development occurs in the thymus and is critically dependent on productive TCRβ rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (β checkpoint), which is uniquely permissive for V-DJβ recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRβ rearrangement and pre-TCR expression enforces suppression of TCRβ rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRβ product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the β checkpoint, would suppress all V-DJβ rearrangement. We previously reported that premature CD28 signaling in murine CD4(-)CD8(-) (DN) thymocytes supports differentiation of CD4(+)CD8(+) (DP) cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRβ rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.